23 research outputs found
Funciones de hCLE en asociación con DDX1, HSPC117 y FAM98B en la modulación de la traducción de proteínas, el reconocimiento de estructuras cap y su implicación en procesos neuronales
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 20-10-2016Esta tesis tiene embargado el acceso al texto completo hasta el 20-04-2018hCLE/C14orf166/CGI/99! es! una! proteína! nuclear! y! citoplasmática! que! forma!
complejos! multiproteícos.! En! ambos! compartimentos! se! asocia! con! las! proteínas! DDX1,!
HSPC117! y! FAM98B,! y! transporta! RNAs! que! codifican! mayoritariamente! proteínas!
relacionadas!con!la!traducción!de!RNAs!y!el!metabolismo!energético,!proteínas!de!función!
neuronal,!canales!iónicos!y!proteínas!de!transporte!vesicular.!hCLE!se!mueve!entre!el!núcleo!
y!el!citoplasma!y!su!reentrada!al!núcleo!es!dependiente!de!transcripción!activa,!siendo!esta!
dependencia!!compartida!por!DDX1,!HSPC117!y!FAM98B.!
hCLE! purificado!une!m7GTP,! un! análogo!de! la!estructura!5’! cap!de! los!mRNAs,!en!
células! HEK293T,! en! células! neuronales! y! en! extractos! de! cerebro! de! rata.! Ésta! unión! se!
produce!con!mayor!afinidad!y!especificidad!que!la!del!factor!citoplasmático!eIF4E,!requerido!
para! la! iniciación! de! la! traducción! de! los!mRNAs.! Además! la! unión! de! hCLE! a!m7GTP! es!
modulada!positivamente!por!DDX1/HSPC117/FAM98B.!!
El! silenciamiento! de! hCLE! reduce! la! acumulación! de! sus! proteínas! asociadas! y! la!
traducción!de!los!mRNAs!sin!afectar!su!transporte!o!su!estabilidad.!El!análisis!de!la!función!
neuronal! de! hCLE!muestra! que! está! presente! en! sinapsis! y! enriquecida! en! sinaptosomas,!
localización! no! compartida! con! sus! proteínas! asociadas,! lo! que!modula! negativamente! su!
capacidad!para!unirse!al!5’!cap!en!este!compartimento!subcelular.!!
En! conjunto! los! datos! sugieren! que! hCLE/DDX1/HSPC117/FAM98B! están! formando!
un! complejo! de! movimiento! núcleo/citoplasmático,! transportando! mRNAs! bloqueados!
traduccionalmente!hasta!su!destino!final.!En!respuesta!a!señales!específicas!el!complejo!de!
hCLE!se!remodelaría!perdiendo!hCLE!la!capacidad!de!unión!a!estructuras!cap,!permitiendo!la!
traducción!de!los!mRNAs!asociados!requeridos!y!cooperando!en!los!mecanismos!locales!de! traducción.!hCLE/C14orf166/CGI/99!is!a!nuclear!and!cytoplasmic!protein!that!forms!multiprotein!
complexes.! In! both! compartments! it! associates! with! DDX1,! HSPC117! and! FAM98B,! and!
transports! RNAs! that! mainly! encode! proteins! related! with! RNA! translation! and! energy!
metabolism,!neuronal! function,! ion! channels!and! vesicle! transport.!hCLE! shuttles!between!
nucleus! and! cytoplasm! and! the! re/entry! to! the!nucleus!depends!on! active! transcription,! a!
behavior!shared!with!its!interactors!DDX1,!HSPC117!and!FAM98B.!
In! HEK293T! cells,! neuronal! primary! cultures! and! brain! tissues,! endogenous! hCLE!
binds!m7GTP,!a!5’!cap!RNA!structure!analog,!with!higher!affinity!and!specificity!than!eIF4E’s,!
a!factor!required!for!mRNA’s!translation!initiation.!Purified!hCLE!also!binds!m7GTP,!and!this!
binding!capacity!is!positively!modulated!by!DDX1/HSPC117/FAM98B.!!
hCLE’s!silencing!decreases!the!accumulation!of!the!associated!proteins!and!inhibits!
overall!mRNA!translation,!not!affecting!mRNA!transport!or!stability.!On!the!other!hand,!the!
analysis!of!hCLE’s!neuronal!function!shows!that!hCLE!is!present!in!synapses!and!enriched!in!
synaptosomes,!whereas!the!accompanied!DDX1,!HSPC117!and!FAM98B!proteins!are!absent.!
Consequently,! synaptosomal! hCLE! looses! the! 5’! cap! binding! activity! in! this! cellular!
compartment.!!
Taking!altogether,!the!data!suggest!that!hCLE/DDX1/HSPC117/FAM98B!are!forming!a!
shuttling!complex!that!transports!mRNAs!in!a!translationally!repressed!way!from!the!nucleus!
to! their! final! destination! in! the! cytoplasm.! In! response! to! specific! signals,! hCLE’s! complex!
remodeling! could! be! triggered,! loosing! hCLE! its! capacity! to! bind! cap! structures! and! thus,!
allowing! the! cap/dependent! translation! of! cargo! mRNAs! in! the! place! where! they! are!
required,!cooperating!with!local!translation!mechanisms
hCLE/RTRAF-HSPC117-DDX1-FAM98B: A New Cap-Binding Complex That Activates mRNA Translation
hCLE/C14orf166/RTRAF, DDX1, and HSPC117 are components of cytoplasmic mRNA-transporting granules kinesin-associated in dendrites. They have also been found in cytoplasmic ribosome-containing RNA granules that transport specific mRNAs halted for translation until specific neuronal signals renders them accessible to the translation machinery. hCLE associates to DDX1, HSPC117, and FAM98B in HEK293T cells and all four proteins bind to cap analog-containing resins. Competition and elution experiments indicate that binding of hCLE complex to cap resins is independent of eIF4E; the cap-binding factor needed for translation. Purified hCLE free of its associated proteins binds cap with low affinity suggesting that its interacting proteins modulate its cap association. hCLE silencing reduces hCLE accumulation and that of its interacting proteins and decreases mRNA translation. hCLE-associated RNAs have been isolated and sequenced; RNAs involved in mRNA translation are specifically associated. The data suggest that RNA granules may co-transport RNAs encoding proteins involved in specific functions together with RNAs that encode proteins needed for the translation of these specific RNAs and indicate an important role for hCLE modulating mRNA translation
Astrocyte GluN2C NMDA receptors control basal synaptic strengths of hippocampal CA1 pyramidal neurons in the stratum radiatum
Experience-dependent plasticity is a key feature of brain synapses for which neuronal N-Methyl-D-Aspartate receptors (NMDARs) play a major role, from developmental circuit refinement to learning and memory. Astrocytes also express NMDARs, although their exact function has remained controversial. Here, we identify in mouse hippocampus, a circuit function for GluN2C NMDAR, a subtype highly expressed in astrocytes, in layer-specific tuning of synaptic strengths in CA1 pyramidal neurons. Interfering with astrocyte NMDAR or GluN2C NMDAR activity reduces the range of presynaptic strength distribution specifically in the stratum radiatum inputs without an appreciable change in the mean presynaptic strength. Mathematical modeling shows that narrowing of the width of presynaptic release probability distribution compromises the expression of long-term synaptic plasticity. Our findings suggest a novel feedback signaling system that uses astrocyte GluN2C NMDARs to adjust basal synaptic weight distribution of Schaffer collateral inputs, which in turn impacts computations performed by the CA1 pyramidal neuron
HCLE/RTRAF-HSPC117-DDx1-FAM98B: A new cap-binding complex that activates mRNA translation
© 2019 Pazo, Pérez-González, Oliveros, Huarte, Chavez and Nieto.hCLE/C14orf166/RTRAF, DDX1, and HSPC117 are components of cytoplasmic mRNA-transporting granules kinesin-associated in dendrites. They have also been found in cytoplasmic ribosome-containing RNA granules that transport specific mRNAs halted for translation until specific neuronal signals renders them accessible to the translation machinery. hCLE associates to DDX1, HSPC117, and FAM98B in HEK293T cells and all four proteins bind to cap analog-containing resins. Competition and elution experiments indicate that binding of hCLE complex to cap resins is independent of eIF4E; the cap-binding factor needed for translation. Purified hCLE free of its associated proteins binds cap with low affinity suggesting that its interacting proteins modulate its cap association. hCLE silencing reduces hCLE accumulation and that of its interacting proteins and decreases mRNA translation. hCLE-associated RNAs have been isolated and sequenced; RNAs involved in mRNA translation are specifically associated. The data suggest that RNA granules may co-transport RNAs encoding proteins involved in specific functions together with RNAs that encode proteins needed for the translation of these specific RNAs and indicate an important role for hCLE modulating mRNA translation.This work was supported by the Spanish Ministry of Economy and Competitiveness (BFU2014-57797; AEI/FEDER, UE MINECO) and Ciber de Enfermedades Respiratorias (CIBERES)
Butanolysis of 4-methylbenzenediazonium ions in binary n-BuOH/H2O mixtures and in n-BuOH/SDS/H2O reverse micelles. Effects of solvent composition, acidity and temperature on the switch between heterolytic and homolytic dediazoniation mechanisms
hCLE/C14orf166 Associates with DDX1-HSPC117-FAM98B in a Novel Transcription-Dependent Shuttling RNA-Transporting Complex
<div><p>hCLE/C14orf166 is a nuclear and cytoplasmic protein that interacts with the RNAP II, modulates nuclear RNA metabolism and is present in cytoplasmic RNA granules involved in localized translation. Here we have studied whether hCLE shares common interactors in the nucleus and the cytosol, which could shed light on its participation in the sequential phases of RNA metabolism. Nuclear and cytoplasmic purified hCLE-associated factors were identified and proteins involved in mRNA metabolism, motor-related proteins, cytoskeletal and translation-related factors were found. Purified hCLE complexes also contain RNAs and as expected some hCLE-interacting proteins (DDX1, HSPC117, FAM98B) were found both in the nucleus and the cytoplasm. Moreover, endogenous hCLE fractionates in protein complexes together with DDX1, HSPC117 and FAM98B and silencing of hCLE down-regulates their nuclear and cytosolic accumulation levels. Using a photoactivatable hCLE-GFP protein, nuclear import and export of hCLE was observed indicating that hCLE is a shuttling protein. Interestingly, hCLE nuclear import required active transcription, as did the import of DDX1, HSPC117 and FAM98B proteins. The data indicate that hCLE probably as a complex with DDX1, HSPC117 and FAM98B shuttles between the nucleus and the cytoplasm transporting RNAs suggesting that this complex has a prominent role on nuclear and cytoplasmic RNA fate.</p></div
Nuclear and cytosolic hCLE-associated proteins.
<p>Table includes proteins where at least 2 non-redundant peptides were identified, with a FDR <1% at peptide level that are present exclusively in hCLE purified samples or have at least 3-fold enrichment of total number of confidently identified peptides in hCLE purified fractions compared to the control (ctr.) samples (marked with asterisk). Their molecular weight, the total number of confidently identified peptides and the number of non-redundant ones, as well as the protein Mascot scores, is shown.</p
hCLE forms dimers that are resistant to denaturing conditions.
<p>(A); Western blot against hCLE of total extracts of HEK293T cells using denaturing (left) or native conditions (right). (B); Diagram showing the scheme of the fusion protein hCLE-TAP with its tags for affinity purification. (C); left, HEK293T cells were transfected with the hCLE-TAP expressing plasmid and total cell extract was used for affinity purification, a sample of the purified protein was analyzed by silver staining in SDS-PAGE gels. Right; The hCLE-CBC purified protein was dialyzed and analyzed in non-denaturing gels followed by Western blot detection. (D). The hCLE-CBD purified protein was subjected to silver staining and bands corresponding to different sizes were excised and analyzed by MS-MS technique. The lines denote hCLE identification by MS-MS technique. (*), denotes hCLE monomer and (**) hCLE dimer.</p
Nuclear and cytosolic hCLE-associated proteins.
<p>Table includes proteins where at least 2 non-redundant peptides were identified, with a FDR <1% at peptide level that are present exclusively in hCLE purified samples or have at least 3-fold enrichment of total number of confidently identified peptides in hCLE purified fractions compared to the control (ctr.) samples (marked with asterisk). Their molecular weight, the total number of confidently identified peptides and the number of non-redundant ones, as well as the protein Mascot scores, is shown.</p
hCLE modulates the expression of hCLE-interacting proteins.
<p>(A); HEK293T cells were infected with lentiviruses expressing a control sequence (Ct) or specific sequences for hCLE silencing (siCLE.1 and si-CLE.2) and 4 days post-infection total cell extracts were used for Western blot against the indicated proteins. (B); HEK293T cells were infected with lentiviruses expressing a control sequence (Ct) or a specific sequence for hCLE silencing (siCLE.1). 5 days post-infection, the cells were left untransfected (-) or transfected (+) with a plasmid expressing a hCLE gene with 3 silent mutations that avoids its silencing (ns.hCLE). 48<b> </b>h post-transfection, nuclear and cytoplasmic fractions were prepared and used for Western blot detection of the indicated proteins.</p